PROJECT SUMMARY/ABSTRACT Obesity, characterized by excessive adiposity, is a risk factor for many metabolic disorders, such as Type 2 diabetes mellitus (T2D). Numerous studies have shown that adipose tissue (AT) distribution may be a greater predictor of metabolic health. Upper-body AT [subcutaneous abdominal (scABD) and visceral (VAT)] is commonly associated with complications of obesity, while lower-body AT [subcutaneous gluteal and femoral (scFEM)] may be protective. The opposing associations of adipose distribution with health risk are likely attributed to inherent depot differences. However, the factors and mechanisms that govern AT depot development and expansion are not well understood. Adipose expansion occurs by hypertrophy (increase in size) and/or hyperplasia (increase in number) of adipocytes. Studies suggest that adipocyte cellularity may be a metabolic determinant, as hypertrophy (enlargement) of adipocytes is associated with obesity-related co- morbidities. Contrary to the routine use of the mean fat cell size, the assessment of different adipocyte size distributions provides a better measure of adipose cell morphology. Multisizer Coulter Counter analysis of osmium-fixed AT generates a bimodal distribution of fat cell sizes, including a population of large and small adipocytes. Limited studies using this method report that hypertrophy of large scABD adipocytes, as well as a greater proportion of small scABD adipocytes are correlated with increased VAT, insulin resistance, and T2D. However, no study has assessed differences in fat cell size distributions between the scABD and the scFEM depots or the association of depot-specific cellularity and metabolic markers. The research objective for the proposed project is to collect and analyze human samples and physiological data from an on-going clinical study [?Cellular Dynamics of Fat Distribution? (R01-DK090607); PI: Ravussin, E)] to investigate how variations in scABD and scFEM adipocyte size populations are related to VAT adiposity, insulin sensitivity, and adipokine (gp130 cytokine) production in women with obesity. The hypotheses are that increased diameter of large scABD adipocytes and higher proportion of small scABD adipocytes will positively correlate with VAT mass, impaired glucose tolerance, and higher production of the gp130 cytokines, cardiotrophin-1 and interleukin-6; while smaller diameter of large scFEM adipocytes and smaller proportion of small scFEM adipocytes will negatively correlate with VAT mass, impaired glucose tolerance, and gp130 cytokine production. The data generated in this research project will set the stage for future R01-level analyses to investigate mechanisms that control regional AT growth and distribution, which is the PI's long-term research goal. Collectively, these and other findings may yield important pharmacological targets for the regulation of body weight and fat distribution.